Treatment of emphysema with retinoic acid or other retinoids by inducing formation of gas-exchange units (alveoli)

ABSTRACT

This invention relates to the use of retinoic acid, its esters and analogues thereof, for treatment of emphysema. The method comprises administration of a composition containing an effective amount of a retinoic acid, or an ester or an analoge thereof, to induce alveolar formation.

This work was supported in part by the national Heart, Lung and BloodInstitute grants HL-37666 and HL-20366. Certain rights in the governmentof the United States apply to this invention.

This application takes priority from U.S. Provisional Patent Application60/052,791, filed Jul. 8, 1997.

FIELD OF THE INVENTION

This invention relates to the treatment of emphysema using retinoicacid, their esters and analogues thereof.

BACKGROUND OF THE INVENTION

Pulmonary emphysema is a common disease in which destruction of thelung's gas-exchange structures (alveoli) leads to inadequateoxygenation, disability and, frequently, death. Lung transplantation haspreviously provided the only means of remediation.

Alveoli are formed by the developmentally regulated subdivision ofsaccules that constitute the gas-exchange region of the immature lung.The molecular signals responsible for the formation of septa and fortheir spacing are poorly understood. However, in the rat retinoids mayplay a key regulatory role. Fibroblasts rich in vitamin A (retinol)storage granules occupy a large fraction of the alveolar wall when septaare being formed. During the same period, the concentration ofcellular-retinol binding protein I, cellular retinoic acid-bindingProtein I, and nuclear retinoic acid receptor-τ mRNA peak in the lung.Treatment with dexamethasone, a glucocorticosteroid hormone, preventsseptation in a seemingly irrevocable fashion, and diminishes theexpression in the lung of cellular retinol-binding protein and retinoicacid receptor-β mRNA.

The use of all-trans retinoic acid for treatment of diseases relating togrowth and tissue maintenance has previously been known. Retinoic acidreceptors belong to a family of nuclear receptors that includesreceptors for steroids, thyroid hormone, and calcitriol. It haspreviously been disclosed that retinoic acid prevents inhibition ofalveoli in rats arising from exposure to dexamethasone. (Am. J. Physiol.270: L305-L310 (1996)). There is no teaching therein regarding use ofretinoic acid for treatment of emphysema.

U.S. Pat. No. 3,171,781 teaches use of vitamin A and guaiacol fortreatment of contagious "air sac disease" in fowl. It is not clear whatthe pathology or nature of the infectious agent of the disease conditionmight have been. There is no teaching therein regarding retinoic acid ortreatment of emphysema.

U.S. Pat. No. 4,606,920 teaches use of vitamins A and C for treatment ofinflammatory changes in the bronchial mucosa. There is no teaching seentherein regarding treatment of diseases involving the alveoli.

U.S. Pat. No. 5,534,261, which is incorporated herein in its entirety asthough fully copied herein, teaches use of retinoic acid to preventformation of adhesion between organ surfaces in body cavities,especially in the peritoneal cavity. No teaching of use for treatment ofemphysema is seen therein.

U.S. Pat. No. 5,556,611, which is incorporated herein in its entirety asthough fully copied herein, teaches use of retinoic acid and estersthereof in the form of aerosols for treatment of mucosal diseases.Diseases to be treated by use of such aerosols include bronchialcarcinoma, acute and chronic bronchitis, acute and chronic functionaldisturbances due to impairment of the trachealbronchial epitheliumfollowing inhalation of dusts and gases, bronchopulmonary dysplasia ofnewborns and carthagena syndrome. There is no suggestion therein thatretinoic acid, its esters or analogues thereof can be administered fortreatment of emphysema, which arises from destruction of the lungalveoli.

DESCRIPTION OF THE INVENTION

This invention relates to the use of retinoic acid, its esters andanalogues thereof for treatment of emphysema. Retinoic acid and itsanalogues are lipophilic compounds and may be administered by any meansknown in the art for systemic administration of lipophilic medicinals,including oral and parenteral administration. The method comprisesadministration of a composition containing an alveoli formation-inducingeffective amount of a retinoic acid, its esters and analogues ofretinoic acid.

Materials and Methods:

Production of Emphysema and Treatment with All-trans Retinoic Acid.Porcine pancreas elastase (2.0 units·g⁻¹ body mass) or an equal volumeof saline was instilled into the trachea of anesthetized adult maleSprague-Dawley rats, which were killed 25 days later. Other rats weretreated with saline or elastase as just described and used twenty-fivedays later to form three groups. Rats initially treated with saline(Group 1), and some rats initially treated with elastase (Group 2), werebegun on daily intraperitoneal injections of cottonseed oil containingall-trans retinoic acid. Other rats initially treated with elastase werebegun on daily intraperitoneal injections of all-trans retinoic acid(500 μg·kg⁻¹) dissolved in cottonseed oil (Group 3). Rats were treateddaily for 12 days and killed on day 13.

Fixation, Tissue Sampling, and Tissue Preparation. Rats wereanesthetized with xylazine (˜10 mg·kg⁻¹) and ketamine (˜75 mg·kg⁻¹) andkilled by cutting the abdominal aorta. Cold 2.5% glutaraldehyde in 0.1 Msodium cacodylate, pH 7.4, was infused into the trachea at atranspulmonary pressure of 20 cm H₂ O. The trachea was ligated, lungswere removed from the thorax, and fixation was continued for 2 h at 0-4°C. Lung volume was measured by volume displacement. Lungs were cut intoblocks; blocks were selected for study using a systematic samplingtechnique. Selected blocks were processed further; we corrected forlinear shrinkage and volume changes that occur during postfixation,dehydration, and embedding.

Alveolar airspace was distinguished from alveolar duct airspace byanalysis of serially sectioned lung. The selector method, which allowsstructures to be selected for analysis based on number rather than onsize, shape, or orientation, was used to choose alveoli for analysis.The volume of an alveolus was estimated by the point-sampled interceptsmethod (for references)⁵. The volume of an individual alveolus wascalculated as previously described. The number of alveoli per lung wascalculated using the identity ##EQU1## where VL is lung volume (measuredby water displacement), Vva is the volume density of alveolar airspace,and va is the mean alveolar volume. Vva and Sa of the gas-exchangeregion were determined by point and intersection counting.

Statistical Methods. For each parameter measured or calculated frommeasurements, values for individual animals were averaged perexperimental group, and the SE was calculated. The significance of thedifference between two groups was obtained using the Mann-Whitney test.The Kruskal-Wallis test was used when more than two groups were comparedand the Mann-Whitney test was then used to compare two populations at atime; the Bonferroni adjustment was used to adjust the significancelevel to the number of tests performed.

Results:

Treatment with retinoic acid reversed the anatomical characteristics andincreased lung volume of elastase-induced emphysema in rats.

In rats killed 25 days after the intratracheal instillation of 0.15 MNaCl (saline) the distance between alveolar walls (Lm) was 71±1.9 μm andalveolar surface area (Sa) 4952±259 cm² (mean±SE, N=3). Twenty-five daysafter the instillation of elastase (N=5) Lm was 93±7 μm and Sa 3992±118cm² (P<0.03 between groups for the same parameter). The larger Lm andsmaller Sa in elastase-treated rats indicate their lungs wereemphysematous. Emphysema produced by elastase becomes progressivelyworse for 1-2 months without spontaneous recovery.

Lung volume was 18% greater in emphysematous rats treated withcottonseed oil, the diluent for RA, (elastase-oil rats) than in ratsgiven saline followed by cottonseed oil (saline-oil rats) or in ratsgiven elastase then RA (elastase-RA rats) (Table 1). Because body masswas the same in all groups (Table 1), and lung volume is proportional tobody mass, the larger lungs of elastase-oil rats reflect diminishedelastic recoil, a characteristic feature of experimental and humanemphysema, rather than lung growth. This effect of elastase on lungvolume was completely reversed by treatment with RA (Table 1).

Distance between alveolar walls (Lm), and the volume of an averagealveolus (va), were higher and alveolar number (Na) lower inelastase-oil rats than in saline-oil rats (Table 1). After correctionfor overexpansion of the lung in elastase-oil rats, Lm and va remainedlarger (P<0.025) than in rats of either other group. Sa was notsignificantly diminished in elastase-oil rats despite the presence of45% fewer alveoli because the low elastic recoil allowed overexpansionof the lung. However, Sa per lung volume, a cardinal feature ofemphysema², was diminished in elastase-oil rats (Table 1). In ratstreated with RAva, Na, and volume-corrected Sa were the same as in ratsnot made emphysematous (saline-oil) and were significantly differentfrom the values in elastase-oil rats (Table 1). In saline-oil,elastase-oil, and elastase-RA rats the percentage of alveoli with avolume ≧10×10⁴ μm³ (chosen arbitrarily) was, respectively, 20, 64, and33. These quantitative differences in architecture of the lung'sgas-exchange region were appreciated on histological lung sections.

Although diminished recoil allows lung volume to increase, the fairlyrigid bony thorax of human adults imposes constraints upon lungenlargement and hence upon the degree to which Sa can be augmented byoverexpansion of the lung. In human emphysema the presence of largebullae can also limit the extent to which expansion can increase Sa.Therefore, to increase Sa for therapeutic purposes, a pharmacologicalagent should ideally induce the formation of alveoli with a highsurface-to-volume ratio. Retinoic acid (RA) does that. More numerousalveoli in elastase-RA were found than in elastase-oil rats and this wasbrought about by the generation of smaller alveoli, i.e. alveoli with ahigh surface-to-volume ratio, rather than by increasing lung volume(Table 1). That the frequency-distribution of alveoli revealed manyfewer large alveoli in elastase-RA rats than in elastase-oil rats,strongly suggests treatment with RA induced the formation of septa fromthe walls of the large alveoli that resulted from prior destruction ofalveolar walls by elastase. The lower lung volume in elastase-RAcompared to elastase-oil rats indicates lung recoil increased in ratstreated with RA.

                  TABLE I                                                         ______________________________________                                        Body weight, lung volume, alveolar size and number and                          surface area in mammals:                                                               Treatments                                                         Parameters Saline-oil Elastase-oil                                                                             Elastase-RA                                  ______________________________________                                        Body weight                                                                               410 ± 11 (6)                                                                          403 ± 7 (7)                                                                           404 ± 5 (7)                                Lung volume, cm.sup.3  10.0 ± 0.5 (6)  11.8 ± 0.5 (7)  10.1 ±                                       0.3 (7)                                        Lung vol, cm.sup.3 · kg.sup.-1  24.3 ± 0.7 (6)  28.8 ±                                        1.4 (7)  24.9 ± 0.6 (7)                     Lm, μm  74 ± 3 (6)  96 ± 5 (7)  67 ± 2 (7)                        va, μm.sup.3 × 10.sup.-4   7.9 ± 0.5 (5)  19.1 ± 1.6 (5)                                        9.3 ± 0.9 (5)                             Na ×10.sup.-6  58.8 ± 7.0 (5)  30.2 ± 2.1 (5)  55.4 ± 36                                      (5)                                            Na 10.sup.-6 · kg.sup.-1  143 ± 21.1 (5)  75.3 ± 4.9 (5)                                       136 ± 9.9 (5)                              Sa, cm.sup.2 4434 ± 251 (6) 4222 ± 90 (7) 4939 ± 136 (7)                                             Sa.lung vol..sup.-1, cm.sup.-1  444                                          ± 12 (6)  362 ± 16 (7)  492 ±                                        10 (7)                                       ______________________________________                                    

Mean±SE given. Figures in parentheses indicate the number of rats. vadenotes the volume of an average alveolus, Na the number of alveoli perrat, Sa the alveolar surface area, and Lm, distance between alveolarwalls.

Treatment with retinoic acid caused a marked reduction in the number oflarge alveoli in rats previously treated with elastase. It was foundthat treatment with retinoic acid abrogated elastase-induced emphysema.The airspaces were larger and less numerous in lungs of rats whoreceived only the cottonseed oil. Treatment with retinoic acid returnedthe lung's morphology to that present in rats previously notemphysematous.

As indicated, the active agents of the invention may be administered inany manner used to administer lipophilic agents, and will depend on themode of administration. Solvents for lipophilic compounds known in theart include (but are not limited to) glycols such as polypropyleneglycol, polyethylene glycol, oils and cyclodextrins, especially theintrinsically amorphous cyclodextrins. Other vehicles that should beconsidered include fatty acid esters of polyoxyethylene sorbatan(Tweens) or sorbitan (Spans) used to prepare oil-in-water emulsions. Theactive agents, retinoic acids and esters or analogues thereof may alsobe administered as liposomes. The active agents may be administered indepo agents such as oils. In the instant examples, cotton oil was used.

The therapeutic compositions may also be administered as an inhalant vianebulizer.

Compositions may be administered by any means that will result in uptakeinto the blood stream, including subcutaneous, intramuscular andintravenous administration. Cyclodextrin inclusion complexes may beadministered sublingually or buccally.

A patch for the administration of the active agents may be formulated asadhesive patches containing the drug. For example, the patch may be adiscoid in which a pressure-sensitive silicone adhesive matrixcontaining the active agent may be covered with a non-permeable backing.The discoid may either contain the active agent in the adhesive or mayhave attached thereto a support made of material such as polyurethanefoam or gauze that will hold the active agent. Before use, the materialcontaining the active agent would be covered to protect the patch.

The dosage required will depend on the size, age and condition of thepatient. Dosage of about 0.1 μg/kg to 1000 μg/kg would be appropriate,with the higher dosage per kg being appropriate in small mammals and thelower dosage per kg of about 0.1 μg/kg to 1000 μg/kg being appropriatein larger mammals such as humans.

EXAMPLE 1

A patch composed of trilaminate of an adhesive matrix sandwiched betweena non-permeable backing and a protective covering layer is prepared inthe following manner:

To a pressure-sensitive silicone adhesive composition BIOPSA™ Q7-2920(Dow Corning Corp., Midland, Michigan, U.S.A.) in cyclohexane (50% w/v)is added sufficient retinoic acid to provide a 0.5% retinoic acidcomposition. The adhesive is applied to a polyester film to provide insuccessive layers to provide about 2 mg of active agent per cm². Thefilm containing the adhesive is then made into patches of 10 cm². Forpatches would be covered with a protective layer to be removed beforeapplication of the patch. Patches may be prepared containing permeationenhancers such as cyclodextrin, butylated hydroxyanisole, or butylatedhydroxytoluene. When the patches are to be applied to thin or abradedskin, there is little need to add a permeation enhancer.

EXAMPLE 2

    ______________________________________                                        Ingredient               w/w %                                                ______________________________________                                        ethyl ester of retinoic acid                                                                           20 μg                                               Propylene glycol  .2 ml                                                       Saline added to total volume of  2 ml                                       ______________________________________                                    

EXAMPLE 3

Saturated aqueous solutions of the sparingly soluble retinoic acid weremade by stirring an excess of the RA with water with an aqueous solution(5% weight by weight) of dihydroxypropylcyclodextrin to formcyclodextrin inclusion complexes. (The stirring step required at leastone day.) The concentration of the drug in a filtered solution wassubsequently determined by spectrophotometry.

The resulting cyclodextrin inclusion complex was dissolved in saline toprovide 10 μg retinoic acid for administration sublingually, buccally orby nebulizer.

In order to obtain more prolonged action, it is possible to administeran ester of the retinoic acid. Examples of such esters are the alkylesters such as methyl, ethyl, propyl and butyl esters. The estermoieties may also have cyclized or aryl groups such as benzyl,hydroxybenzyl and cyclohexyl esters.

What we claim is:
 1. A method of treating emphysema by increasing theformation of alveoli in a mammal comprising administration of acomposition containing, an effective amount of a retinoic acid or anester or analog of retinoic acid, in a pharmaceutically acceptablecarrier.
 2. A method of claim 1 wherein the carrier is a patch.
 3. Amethod of claim 1 wherein the mammal is a human.
 4. A method of claim 1wherein the composition is administered parenterally.
 5. A method ofclaim 1 wherein the composition is administered either buccally,sublingually or by nebulizer.